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Biomedical subjects

Tetsuo Nagano

Publications and source records attributed to Tetsuo Nagano.

At least 19 recordsLinked to original sources
PubMed2006

Modulation of luminescence intensity of lanthanide complexes by photoinduced electron transfer and its application to a long-lived protease probe.

Luminescent lanthanide complexes (Tb(3+), Eu(3+), etc.) have excellent properties for biological applications, including extraordinarily long lifetimes and large Stokes shifts. However, there have been few reports of lanthanide-based functional probes, because of the difficulty in designing suitable complexes with a luminescent on/off switch. Here, we have synthesized a series of complexes which consist of three moieties: a lanthanide chelate, an antenna, and a luminescence off/on switch. The antenna is an aromatic ring which absorbs light and transmits its energy to the metal, and the switch is a benzene derivative with a different HOMO level. If the HOMO level is higher than a certain threshold, the complex emits no luminescence at all, which indicates that the lanthanide luminescence can be modulated by photoinduced electron transfer (PeT) from the switch to the sensitizer. This approach to control lanthanide luminescence makes possible the rational design of functional lanthanide complexes, in which the luminescence property is altered by a biological reaction. To exemplify the utility of our approach to the design of lanthanide complexes with a switch, we have developed a novel protease probe, which undergoes a significant change in luminescence intensity upon enzymatic cleavage of the substrate peptide. This probe, combined with time-resolved measurements, was confirmed in model experiments to be useful for the screening of inhibitors, as well as for clinical diagnosis.

Chemistry, Clinical↗
PubMed2006

Development of a ratiometric fluorescent zinc ion probe in near-infrared region, based on tricarbocyanine chromophore.

Novel ratiometric fluorescent probes for Zn2+ in the near-infrared region, based on a tricarbocyanine chromophore, have been designed, synthesized, and evaluated. Upon addition of Zn2+, a 44 nm red shift of the absorption maximum was observed, which indicates that this probe could work as a ratiometric probe for Zn2+. This change is due to the difference in the electron-donating ability of the amine substituent before and after reaction with Zn2+. This fluorescence modulation of amine-substituted tricarbocyanines should be applicable to dual-wavelength measurement of various biomolecules or enzyme activities.

Alkanesulfonates↗
PubMed2006

Inhibition of presynaptic activity by zinc released from mossy fiber terminals during tetanic stimulation.

Zinc exists in high densities in the giant boutons of hippocampal mossy fibers. On the basis of the evidence that zinc decreases extracellular glutamate concentration in the hippocampus, the presynaptic action of zinc released from mossy fibers during high-frequency (tetanic) stimulation was examined using hippocampal slices. The increase in zinc-specific fluorescent signals was observed in both extracellular and intracellular compartments in the mossy fiber terminals during the delivery of tetanic stimuli (100 Hz, 1 sec) to the dentate granule cell layer, suggesting that zinc released from mossy fibers is immediately retaken up by mossy fibers. When mossy fiber terminals were preferentially double-stained with zinc and calcium indicators and tetanic stimuli (100 Hz, 1 sec) were delivered to the dentate granule cell layer, the increase in calcium orange signal during the stimulation was enhanced in mossy fiber terminals by addition of CaEDTA, a membrane-impermeable zinc chelator, and was suppressed by addition of zinc. The decrease in FM4-64 signal (vesicular exocytosis) during tetanic stimulation (10 Hz, 180 sec), which induced mossy fiber long-term potentiation, was also enhanced in mossy fiber terminals by addition of CaEDTA and was suppressed by addition of zinc. The present study demonstrates that zinc released from mossy fibers may be a negative-feedback factor against presynaptic activity during tetanic stimulation.

Animals↗
PubMed2005

The detection and quantification of highly reactive oxygen species using the novel HPF fluorescence probe in a rat model of focal cerebral ischemia.

A novel fluorescence probe, 2-[6-(4'-hydroxy) phenoxy-3H-xanthen-3-on-9-yl] benzoic acid (HPF) was used to investigate the generation of highly reactive oxygen species (hROS) under ischemia both in vitro and in vivo. In the in vitro study, HT 22 cells were used to demonstrate that was predominantly detected in the cytoplasm, which coincides with the location of the mitochondria and then its HPF fluorescence gradually increased from 6 to 24 h due to glutamate induced oxidative stress. In the in vivo study, the permanent and transient middle cerebral artery occlusion (MCAO) was induced in rats. Brain slices were incubated in an artificial medium containing HPF. The area of enhanced HPF fluorescence existed in both the ischemic core and the peri-infarct area at 4h after MCAO in both permanent and transient MCAO models. The area extended beyond the boundary of the ischemic damage into biochemically viable tissue. The enhanced fluorescent intensity following transient MCAO was higher than that observed in the permanent MCAO model. Hydroxyl radical scavenger, MCI-186 significantly suppressed the enhanced fluorescence intensity. This study demonstrated that HPF has a high sensitivity and specificity for the detection of hROS in focal cerebral ischemia as well as in a cellular model of oxidative stress.

Animals↗
PubMed2005

Evidence for pH dependent Zn2+influx in K562 erythroleukemia cells: studies using ZnAF-2F fluorescence and 65Zn2+ uptake.

Using both ZnAF-2F (a Zn2+ specific fluorophore) and 65Zn2+, we determined the rate of transporter mediated Zn2+ influx (presumably mediated by the SLC39A1 gene product, protein name hZIP1) under steady state conditions and studied the effects of extracellular acidification. When K562 erythroleukemia cells were placed in Zn2+ containing buffers (1-60 microM), the initial rate of 65Zn2+ accumulation mirrored the apparent rise in free intracellular Zn2+ concentrations sensed by ZnAF-2F. Therefore, newly transported Zn2+ equilibrated with the free intracellular Zn2+ pool sensed by ZnAF-2F. A new steady state with elevated free intracellular Zn2+ was established after about 30 min. An estimate of 11 microM for the Km and 0.203 nmol/mg/s for the Vmax were obtained for Zn2+ influx. 65Zn2+ uptake and ZnAF-2F fluorescent changes were inhibited by extracellular acidification (range tested: pH 8-6, IC50 = pH 6.34). The IC50 for proton effects was close to the pKa for histidine, suggesting conserved histidine residues present in SLC39A1 play a critical role in Zn2+ influx and are involved in the pH effect.

Cation Transport Proteins↗
PubMed2005

Highly efficient and photostable photosensitizer based on BODIPY chromophore.

Photosensitizers are reagents that produce reactive oxygen species upon light illumination and are commonly used to study oxidative stress or for photodynamic therapy. There are many available photosensitizers, but most have limitations, such as low photostability, structural instability, or a limited usable range of solvent conditions. Here, we describe a novel photosensitizer scaffold (2I-BDP) based on the unique characteristics of the BODIPY chromophore (i.e., high extinction coefficient, high photostability, and insensitivity to solvent environment). 2I-BDP shows stronger near-infrared singlet oxygen luminescence emission and higher photostability than the well-known photosensitizer, Rose Bengal. Unlike other photosensitizers, this scaffold is widely applicable under various conditions, including lipophilic and aqueous environments. HeLa cells loaded with 2I-BDP could be photosensitized by light illumination, demonstrating that 2I-BDP is potentially useful as a reagent for cell photosensitization, oxidative stress studies, or PDT.

Boron Compounds↗
PubMed2005

Selective zinc sensor molecules with various affinities for Zn2+, revealing dynamics and regional distribution of synaptically released Zn2+ in hippocampal slices.

We have developed a series of fluorescent Zn(2+) sensor molecules with distinct affinities for Zn(2+), because biological Zn(2+) concentrations vary over a wide range from sub-nanomolar to millimolar. The new sensors have K(d) values in the range of 10(-8)-10(-4) M, compared with 2.7 nM for ZnAF-2. They do not fluoresce in the presence of other biologically important metal ions such as calcium or magnesium, and they can detect Zn(2+) within 100 ms. In cultured cells, the fluorescence intensity of ZnAF-2 was saturated at low Zn(2+) concentration, while that of ZnAF-3 (K(d) = 0.79 muM) was not saturated even at relatively high Zn(2+) concentrations. In hippocampal slices, we measured synaptic release of Zn(2+) in response to high-potassium-induced depolarization. ZnAF-2 showed similar levels of fluorescence increase in dentate gyrus (DG), CA3 and CA1, which were indistinguishable. However, ZnAF-3 showed a fluorescence increase only in DG. Thus, by using a combination of sensor molecules, it was demonstrated for the first time that a higher Zn(2+) concentration is released in DG than in CA3 or CA1 and that we can easily visualize Zn(2+) concentration over a wide range. We believe that the use of various combinations of ZnAF family members will offer unprecedented versatility for fluorescence-microscopic imaging of Zn(2+) in biological applications.

Animals↗
PubMed2005

Evolution of fluorescein as a platform for finely tunable fluorescence probes.

Fluorescence imaging is the most powerful technique currently available for continuous observation of dynamic intracellular processes in living cells. Suitable fluorescence probes are naturally of critical importance for fluorescence imaging, but only a very limited range of biomolecules can currently be visualized because of the lack of flexible design strategies for fluorescence probes. At present, design is largely empirical. Here we show that the carboxylic group of traditional fluorescein dyes, formerly considered indispensable, has been replaced with other substituents, affording various kinds of new fluoresceins. Further, by breaking out of the traditional structure of fluorescein, we developed the first and totally rational design strategy for novel fluorescence probes based on a strict photochemical basis. The value of this approach is exemplified by its application to develop a novel, highly sensitive, and membrane-permeable fluorescence probe for beta-galactosidase, which is the most widely used reporter enzyme.

Biosensing Techniques↗
PubMed2005

Highly sensitive near-infrared fluorescent probes for nitric oxide and their application to isolated organs.

Novel near-infrared (NIR) fluorescent probes for nitric oxide (NO) have been designed, synthesized, and evaluated. Their NIR fluorescence was increased in an NO concentration-dependent manner under physiological conditions, and their reaction efficiency with NO was at least 53 times higher than that of a widely used NO probe, DAF-2. They were confirmed to function in isolated intact rat kidneys. Because NIR light can penetrate deeply into tissues, these probes may have potential for in vivo NO imaging.

Animals↗
PubMed2005

Inhibition of autotaxin by lysophosphatidic acid and sphingosine 1-phosphate.

Autotaxin (ATX) or nucleotide pyrophosphatase/phosphodiesterase 2 (NPP2) is an NPP family member that promotes tumor cell motility, experimental metastasis, and angiogenesis. ATX primarily functions as a lysophospholipase D, generating the lipid mediator lysophosphatidic acid (LPA) from lysophosphatidylcholine. ATX uses a single catalytic site for the hydrolysis of both lipid and non-lipid phosphodiesters, but its regulation is not well understood. Using a new fluorescence resonance energy transfer-based phosphodiesterase sensor that reports ATX activity with high sensitivity, we show here that ATX is potently and specifically inhibited by LPA and sphingosine 1-phosphate (S1P) in a mixed-type manner (Ki approximately 10(-7) M). The homologous ecto-phosphodiesterase NPP1, which lacks lysophospholipase D activity, is insensitive to LPA and S1P. Our results suggest that, by repressing ATX activity, LPA can regulate its own biosynthesis in the extracellular environment, and they reveal a novel role for S1P as an inhibitor of ATX, in addition to its well established role as a receptor ligand.

Allosteric Site↗
PubMed2005

Design and synthesis of zinc-selective chelators for extracellular applications.

Zinc (Zn2+) is found in every cell in human bodies. A few millimolar of free Zn2+ exists in the vesicles of presynaptic neurons in the mammalian brain and is released by synaptic activity or depolarization, modulating the function of certain ion channels and receptors. Although various chemical tools for measuring Zn2+ in biological samples, such as fluorescent probes for Zn2+, have been developed, Zn2+-selective chelators have room to be improved. Research on Zn2+ signals in the brain has traditionally employed several chelators, which have several shortcomings for biological applications. Here we report the design, synthesis, and properties of new membrane-impermeable chelators selective for Zn2+ and describe biological applications in hippocampal slices. As a result, our newly designed chelator revealed the first biological implication that presynaptic Zn2+ can be released in the CA1 region. This confirms the utility of these new chelatotrs as extracellular Zn2+ chelators for biological applications.

Animals↗
PubMed2005

Enhanced excitability of hippocampal mossy fibers and CA3 neurons under dietary zinc deficiency.

On the basis of the evidence that susceptibility to kainate-induced seizures is enhanced by zinc deficiency and that glutamate concentrations in hippocampal extracellular fluid are excessively increased during seizures, excitability of hippocampal mossy fibers and CA3 neurons was examined using hippocampal slices, which were prepare from mice fed a zinc-deficient diet for 4 weeks. The spatio-temporal dynamics of zinc and calcium was monitored using their indicators, membrane-impermeable ZnAF-2 and membrane-permeable fura-2 AM, respectively. When the molecular layer of dentate gyrus was stimulated with 100mM KCl for 1s, the increased percentages of extracellular zinc in the stratum lucidum and CA3 pyramidal cell layer were higher in zinc-deficient mice than in the control mice, implying that glutamate release from the mossy fibers of the dentate granular cells is enhanced by zinc deficiency. Judging from the increased percentages, however, the amount of zinc released was estimated to be less in zinc-deficient mice. On the other hand, the basal calcium concentrations in the stratum lucidum and CA3 pyramidal cell layer detected with fura-2 were higher in zinc-deficient mice than in the control mice, indicating that hippocampal calcium homeostasis is affected by zinc deficiency. Furthermore, the increased percentage of intracellular calcium in the stratum lucidum by stimulation with high K+ was enhanced by the zinc deficiency. The alteration of hippocampal calcium homeostasis seems to enhance excitability of dentate granular cells in zinc deficiency, following by an enhanced excitability of postsynaptic structures in CA3 neurons.

Animals↗
PubMed2004

Rational principles for modulating fluorescence properties of fluorescein.

Rational design strategies based on practical fluorescence modulation mechanisms would enable us to rapidly develop novel fluorescence probes for target molecules. Here, we present a practical and general principle for modulating the fluorescence properties of fluorescein. We hypothesized that (a) the fluorescein molecule can be divided into two moieties, i.e., the xanthene moiety as a fluorophore and the benzene moiety as a fluorescence-controlling moiety, even though there is no obvious linker structure between them, and (b) the fluorescence properties can be modulated via a photoinduced electron transfer (PeT) process from the excited fluorophore to a reducible benzene moiety (donor-excited PeT; d-PeT). To evaluate the relationship between the reduction potential of the benzene moiety and the fluorescence properties, we designed and synthesized various derivatives in which the reduction potential of the benzene moiety was fine tuned by introducing electron-withdrawing groups onto the benzene moiety. Our results clearly show that the fluorescence properties of fluorescein derivatives were indeed finely modulated depending upon the reduction potential of the benzene moiety. This information provides a basis for a practical strategy for rational design of novel functional fluorescence probes.

Drug Design↗
PubMed2004

Development of a zinc ion-selective luminescent lanthanide chemosensor for biological applications.

Detection of chelatable zinc (Zn(2+)) in biological studies has attracted much attention recently, because chelatable Zn(2+) plays important roles in many biological systems. Lanthanide complexes (Eu(3+), Tb(3+), etc.) have excellent spectroscopic properties for biological applications, such as long luminescence lifetimes of the order of milliseconds, a large Stoke's shift of >200 nm, and high water solubility. Herein, we present the design and synthesis of a novel lanthanide sensor molecule, [Eu-7], for detecting Zn(2+). This europium (Eu(3+)) complex employs a quinolyl ligand as both a chromophore and an acceptor for Zn(2+). Upon addition of Zn(2+) to a solution of [Eu-7], the luminescence of Eu(3+) is strongly enhanced, with high selectivity for Zn(2+) over other biologically relevant metal cations. One of the important advantages of [Eu-7] is that this complex can be excited with longer excitation wavelengths (around 340 nm) as compared with previously reported Zn(2+)-sensitive luminescent lamthanide sensors, whose excitation wavelength is at too high an energy level for biological applications. The usefulness of [Eu-7] for monitoring Zn(2+) changes in living HeLa cells was confirmed. This novel Zn(2+)-selective luminescent lanthanide chemosensor [Eu-7]should be an excellent lead compound for the development of a range of novel luminescent lanthanide chemosensors for biological applications.

Biosensing Techniques↗
PubMed2004

AMP-activated protein kinase inhibits angiotensin II-stimulated vascular smooth muscle cell proliferation.

BACKGROUND: AMP-activated protein kinase (AMPK) is a stress-activated protein kinase that works as a metabolic sensor of cellular ATP levels. Here, we investigated whether AMPK signaling has a role in the regulation of the angiotensin II (Ang II)-induced proliferation signal in rat vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: Aminoimidazole-4-carboxamide-1-beta-ribofuranoside (AICAR) activated AMPK in rat VSMCs and inhibited Ang II-induced extracellular signal-regulated kinase 1/2 phosphorylation but not that of p38 MAPK or Akt/PKB. Although Ang II activated AMPK, this activation was significantly inhibited by catalase, N-acetylcysteine, and diphenyleneiodonium chloride, an NADPH oxidase inhibitor. Moreover, the observation that AMPK was activated by H2O2 suggests that AMPK is redox sensitive. The Ang II type 1 receptor antagonist valsartan but not the Ang II type 2 receptor antagonist PD123319 significantly inhibited Ang II-induced AMPK activation, suggesting that Ang II-induced AMPK activation was Ang II type 1 receptor dependent. Whereas 3H-thymidine incorporation by VSMCs treated with Ang II was significantly inhibited when the cells were pretreated with 1 mmol/L AICAR, the inhibition of AMPK by dominant-negative AMPK overexpression augmented Ang II-induced cell proliferation. Subcutaneous injection of AICAR (1 mg/g body weight per day) for 2 weeks suppressed neointimal formation after transluminal mechanical injury of the rat femoral artery. CONCLUSIONS: Our findings indicate that Ang II-induced AMPK activation is synchronized with extracellular signal-regulated kinase signaling and that AMPK works as an inhibitor of the Ang II proliferative pathway. AMPK signaling might serve as a new therapeutic target of vascular remodeling in cardiovascular diseases.

AMP-Activated Protein Kinases↗
PubMed2004

Determination of origin of ephedrine used as precursor for illicit methamphetamine by carbon and nitrogen stable isotope ratio analysis.

The sale of ephedrine, one of the precursors of methamphetamine, is strictly controlled and monitored in various countries to prevent the production of illicit methamphetamine. There are three kinds of production scheme for ephedrine manufacture, and it is very useful for precursor control to investigate the origin of ephedrine used for the synthesis of illicit methamphetamine. By means of stable isotope ratio mass spectrometry (IR-MS), we investigated the origin of ephedrine based on the delta(13)C and delta(15)N values. The various origins of ephedrine (biosynthetic, semisynthetic, or synthetic) could be discriminated clearly by using these values. The delta(15)N values of synthetic ephedrine were more negative than those of ephedrine from other sources. By the repeated distillation of methylamine in our laboratory, we confirmed that this could be due to isotope separation during distillation for the purification of methylamine used for ephedrine synthesis. The values for ephedrine used as the precursor were well-correlated with those for methamphetamine synthesized from it. This drug characterization analysis should be useful to illuminate the origin of the precursors used for clandestine methamphetamine and to trace the diversion of medicinal ephedrine for illicit manufacture of methamphetamine.

Carbon Isotopes↗